The present invention relates to novel monoclonal antibodies against the calcium binding protein S100, the use of these antibodies for the development of immunoassays for determination of total S100 or specific determination of the different isoforms of S100 in serum, plasma, cerebrospinal fluid and other body fluids.
S100 is a 21 kDa protein belonging to the S100/calmodulin/troponin C super family of EF-hand calcium binding proteins (for review see Zimmer et al, Brain Res Bull 37: 417-429, 1995). S100 was originally isolated by Moore and co-workers from human brain, and was originally considered as a brain specific protein (Moore, Biochem Biophys Res Commun 19: 739-744, 1965). There has today been identified 16 members of the S100 family based upon amino acid sequence and functional similarities (Zimmer et al. Brain Res Bull 37: 417-429, 1995).
S100 in brain tissue consists of homo- and heterodimers of xcex1 and xcex2 subunits. The xcex1 and xcex2 subunits show a high degree of sequence and species homology (Isobe et al, Eur J Biochem., 89: 379-388, 1978; Isobe et al. Eur J Biochem. 116: 79-86, 1981). The S100xcex2xcex2 exists in high concentration in glial cells and Schwann cells, while the S100xcex1xcex2 is expressed in glial cells but not in Schwann cells (Isobe et al, J Neurochem 43: 1494-1496, 1984). S100xcex1xcex1 is present in neurones, but is mainly expressed in extra cerebral tissues such as skeletal muscle, kidney and heart (Kato and Kimura, Biochim. Biophys Acta 842:146-152, 1985). Small amounts of S100 is present in different normal human tissues: skin, chondrocytes, spleen, adipose tissue, kidney, heart, skeletal muscle and in different malignant tissues; malignant melanoma, glioma, and soft tissue tumours (Zimmer et al. Brain Res Bull 37: 417-429, 1995).
Determination of S100 in serum and CSF has shown to be useful in the management of patients with brain trauma and to evaluate neurological complications after cardiopulmonary bypass surgery (Michetti et al. J Neurol. Sci. 44: 731-743 1980; Persson et al. Stroke 18: 911-918, 1987; Ingebrightsen et al. J Neurol, Neurosurg. Psych. 59: 103-104, 1995; Westaby et al. Ann Thorac Surg 61: 88-92, 1996). Serological determination of S100 has also been shown to be useful for follow-up and in order to obtain prognostic information in patients with malignant melanoma (Henze et al. Dermatology 194: 208-212, 1997; Buer et al. Br J Cancer, 75: 1373-1376, 1997). Different disorders may be associated with changed total amount of S100xcex1xcex2 and S100xcex2xcex2 or changed ratio between the different isoforms of S100. Therefore accurate methods for determining the total amount of S100xcex1xcex2 and S100xcex2xcex2 as well as for determining the different isoforms of S100xcex1xcex2 and S100xcex2xcex2 are needed.
WO 98/01471 discloses a method for determining the total amount of S100. This method comprises one step with the major drawback that information of the amount of the individual isoforms S100xcex1xcex2 and S100xcex2xcex2 is not given, and the response for S100xcex1xcex2 and S100xcex2xcex2 is not identical, hence the assay according to the invention gives a more accurate determination of total S100 (i.e. the sum of S100xcex1xcex2 and S100xcex2xcex2). The method according to the present invention gives a more accurate determination of the total amount S100 since the S100xcex1xcex2 and S100xcex2xcex2 are determined in two separate steps with specific determination of the isoforms S100xcex1xcex2 and S100xcex2xcex2 in the individual steps. The method according to the present invention also gives accurate information of the concentration of the two isoforms S100xcex1xcex2 and S100xcex2xcex2.
The present invention provides monoclonal antibodies against S100, and the design of immunoassays for determination of the total amount of S100xcex1xcex2 and S100xcex2xcex2 as well as for specific determination of the different isoforms of S100, i.e. specific for either S100xcex1xcex2 or S1000xcex2xcex2.
The invention will be useful for serological determination of total S100 and for determination of the different isoforms of S100 in the management of patients with brain damage, neurodegenerative diseases, and in the follow-up of patients with malignant melanoma, gliomas and certain soft tissue tumours.
The present invention provides novel monoclonal antibodies against S100, and the design of immunoassays for determination of S100 in serum, plasma, cerebrospinal fluid or other body fluids.
The method comprises the establishment of monoclonal antibodies against specific epitopes of S100, the use of these antibodies for the design of immunoassays for determination of total S100, immunoassays specific for S100xcex1xcex2 and immunoassays specific for S100xcex2xcex2.
The immunosassays are designed for, but not restricted to, immunometric (non-competitive) assay format. The antibodies according to the invention may also be used for competitive assay format. The immunoassays employ, but are not restricted to, monoclonal antibodies against specific epitopes of S100xcex2 for catching and detection of S100. According to the invention monoclonal antibodies are preferred, but the assay according to the invention may employ polyclonal antibodies, peptides, proteins, protein or DNA fragments with essentially the same binding specificity as the monoclonal antibodies provided by the invention.
Furthermore the immunoassays employ, but are not restricted to, a solid-phase containing the S100 monoclonal antibodies for the adsorption of the S100 from the sample. The solid-phase may be wells of microtiter plates, plastic tubes, beads, magnetic particles or the like. The solid-phase may be produced from glass, polypropylene, polystyrene, and the like. The catching antibody may be bound to the solid phase by, but is not restricted to, physical adsorption, covalent binding or through biotinxe2x80x94avidin bridges, or the like, using methods generally known to those skilled in the art.
In the present invention a wide variety of labels are suitable for detection of the S100. Enzymes are of particular interest, including hydrolases particularly esterases and glycosidases, and oxidoreductases, particularly peroxidases. Suitable are also radioactive labels, such as 125I, 32P, 14C, 3H and the like, fluorescent compounds such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, lanthanid derivatives, and the like, and chemiluminencers such as luciferin, luminol, acridinium esters and the like.
The assays according to the invention can be performed in any double epitope assay format; either two-step forward sandwich assay, one-step sandwich assay or backward sandwich assay.
In performing the assays according to the present invention, a sample (preferably 1-1000 xcexcL) is incubated in the test receptacle containing a catching anti S100 MAb. In the preferred configuration the sample consists of serum, plasma or cerebrospinal fluid. According to the invention the sample and S100 MAb is incubated under conditions allowing binding of the S100 antigen to the catching S100 MAb. In the preferred configuration the catching antibody is immobilized to the solid-phase through biotinxe2x80x94streptavidin bridges. For the detection of the S100 antigen adsorbed to the solid-phase bound antibody a labeled monoclonal antibody directed against independent epitopes of S100 is added, and incubated in the test receptacle under conditions allowing the antibody to bind to the S100-antibody complex. After washing the label is detected in a svstem suitable for the label used.
The present invention provides antibodies specific for the different isoforms of S100 that are used as catching antibodies and antibodies, whose epitopes are not overlapping the epitopes of said catching antibodies, that are used as detecting antibodies (see Example 2.3b). Depending on the selection of catching and detecting antibodies immunoassays with the preferred specificity may be designed, i.e. assays for specific determination of S100xcex1xcex2, and assays for specific determination of S100xcex2xcex2.
The hybridoma cell lines S10:3, S21:4, S23:2, and S35:2, producing the monoclonal antibodies according to the invention, were deposited at the ECACC (European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, Wiltshire SP4 0JG, UK) and assigned ECACC Accession Nos. 98082619, 98082618, 98082617, and 98082616, respectively. These deposits were made on the Aug. 26, 1998. Also included in the invention are antibodies, antibody fragments or peptides with the same binding specificity as antibodies produced by the hybridoma cell lines S10:3, S35:2, S23:2, and S21:4 as shown by that they inhibit the binding of the antibody in question by  greater than 75% as measured by inhibition studies as outlined in Example 2.3b.